like receptors (TLRs) of the innate immune system contribute to noninfectious inflammatory processes. We employed a murine model of hilar clamping (1 h) with reperfusion times between 15 min and 3 h in TLR4-sufficient (C3H/ OuJ) and TLR4-deficient (C3H/HeJ) anesthetized mice with additional studies in chimeric and myeloid differentiation factor 88 (MyD88)-and TLR4-deficient mice to determine the role of TLR4 in lung ischemia-reperfusion injury. Human pulmonary microvascular endothelial monolayers were subjected to simulated warm ischemia and reperfusion with and without CRX-526, a competitive TLR4 inhibitor. Functional TLR4 solely on pulmonary parenchymal cells, not bone marrow-derived cells, mediates early lung edema following ischemia-reperfusion independent of MyD88. Activation of MAPKs and NF-B was significantly blunted and/or delayed in lungs of TLR4-deficient mice as a consequence of ischemia-reperfusion injury, but edema development appeared to be independent of activation of these signaling pathways. Pretreatment with a competitive TLR4 inhibitor prevented edema in vivo and reduced actin cytoskeletal rearrangement and gap formation in pulmonary microvascular endothelial monolayers subjected to simulated warm ischemia and reperfusion. In addition to its well-accepted role to alter gene transcription, functioning TLR4 on pulmonary parenchymal cells plays a key role in very early and profound pulmonary edema in murine lung ischemiareperfusion injury. This may be due to a novel mechanism: regulation of endothelial cell cytoskeleton affecting microvascular endothelial cell permeability. microvascular permeability; endothelial cell; pulmonary edema ACUTE LUNG INJURY IS A FEATURE of sepsis, systemic inflammatory response, and adult respiratory distress syndrome. Noncardiogenic pulmonary edema and impaired gas exchange are consequences of acute lung injury, irrespective of etiology. The mechanisms causing pulmonary edema due to acute lung injury are not well-understood. Ischemia-reperfusion injury (IRI), a form of acute lung injury occurring immediately following lung transplantation, is a frequent complication causing morbidity and mortality (26). A greater understanding of lung IRI is likely relevant to many types of acute lung injury and thus may benefit not only lung transplant recipients, but also substantial numbers of other patients with lung injury. Such knowledge would also facilitate retrieval of lungs from nonheart-beating cadaver donors for transplant and/or may assist in the salvage of lungs not considered suitable for transplant, thereby reducing the critical shortage of transplantable lungs (8, 11).Reperfusion following an interval of ischemia results in an inflammatory response involving components of the innate immune system, including the complement and coagulation cascades. Both parenchymal and myeloid cells elaborate free radicals, nitric oxide, and pro-and anti-inflammatory cytokines (4, 5). Recently, there has been increasing awareness of the important contribution of the innate immune syst...

Arterial hyperresponsiveness to serotonin (5-hydroxytryptamine, 5-HT) is observed in experimental models and human forms of hypertension. Presently, we test the hypothesis that the 5-HT 2B receptor is up-regulated and necessary for maintaining elevated blood pressure in a rat made hypertensive by the nitric-oxide synthase inhibitor N -nitro-L-arginine (LNNA; 0.5 g/l). After 2 weeks of treatment, thoracic aorta were removed from LNNA hypertensive (systolic blood pressure ϭ 189 Ϯ 5 mm Hg) and sham normotensive rats (121 Ϯ 1 mm Hg), denuded, and mounted into isolated tissue baths for measurement of isometric contraction. In sham tissues, 5-HT-induced contraction was mediated by the 5-HT 2A receptor as evidence by a parallel rightward shift in response to 5-HT by the 5-HT 2A/2C receptor antagonist ketanserin (10 nM) and lack of shift by the 5-HT 2B receptor antagonist 6-methyl-1,2,3,4-tetrahydro-1-[3,4-dimethoxyphenyl)methyl]-9H-pyrido [3,4-b]indole hydrochloride (LY272015) (10 nM). In contrast, LY272015 produced a 4-fold rightward shift to 5-HT in aorta from LNNA hypertensive rats, and blockade by ketanserin did not occur at low concentrations of 5-HT. Maximal contraction to the 5-HT 2B receptor agonist 1-[5-(2-thienylmethoxy)-1H-3-indolyl]propan-2-amine hydrochloride was significantly greater in LNNA hypertensive rats (percentage of phenylephrine contraction in sham ϭ 7 Ϯ 4, and in LNNA ϭ 61 Ϯ 7%). 5-HT 2B receptor protein was present in aortic homogenates from sham and LNNA rats, but the density of 5-HT 2B receptor protein in LNNA homogenates was 300% that in sham. Importantly, the 5-HT 2B receptor antagonist LY272015 reduced blood pressure of the LNNA hypertensive but not the sham normotensive rats. Thus, these data suggest that the up-regulated 5-HT 2B receptor in the LNNA hypertensive rats is physiologically activated to maintain elevated blood pressure.Abnormal vascular responsiveness to contractile agonists is often observed in forms of hypertension, including spontaneous, genetic, and experimental models. In particular, the augmented contractile response to serotonin (5-hydroxytryptamine, 5-HT) in isolated vascular preparations and whole animal studies can be profound (McGregor and Smirk, 1970;Hurley-Myers et al., 1985). In nondiseased conditions, 5-HT causes constriction in most vascular beds, although in coronary vascular tissues 5-HT reacts with endothelial cells to mediate relaxation of the smooth muscle cells (Martin, 1994;Glusa and Roos, 1996). 5-HT is produced in the enterochromaffin cells of the gut and released into the circulation where the majority of the monoamine is enzymatically degraded by the liver in the portal circulation (Vanhoutte, 1991). The small amount of 5-HT remaining is broken down by oxidases in the pulmonary endothelial cells or taken up by platelets. Ultimately, circulating levels of free 5-HT are low (12-150 nM) unless vascular damage or disease is present. If platelets are activated, this can result in a localized release of 5-HT from the dense granules. 5-HT can then ...

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